Vibrio cholerae is a bacterial pathogen endemic to many regions of the world where it is commonly found in the aquatic environment. When water contaminated with V. cholerae is ingested by a human host, the bacteria colonize the small intestine where they produce cholera toxin, resulting in disease. Virulence genes, including cholera toxin, are expressed at a very high level near the epithelium of the small intestine, but not in the lumen. In the lumen, the bacteria encounter high concentrations of bile, a negative regulator of virulence genes. When the bacteria penetrate the mucus layer and approach the epithelium, bile levels decrease and they encounter bicarbonate, a positive regulator of virulence genes. They also meet with rising levels of antimicrobial peptides (APs), which are produced by the intestinal epithelial cells as part of innate immunity. Our laboratory recently found that several cationic APs further elevate virulence gene expression in combination with bicarbonate treatment. Therefore, we hypothesize that V. cholerae uses antimicrobial peptides from intestinal epithelial cells in the human host as a key spatial signal to increase virulence gene expression. The objective of the proposed studies is to better understand the role of this signal in cholera pathogenesis. The objective of this proposal will be accomplished by pursuing two specific aims: 1) Characterize the molecular mechanism(s) by which antimicrobial peptides enhance virulence gene expression; and 2) Determine the consequences of responding to these antimicrobial peptide signals in the pathogenesis of cholera. Completion of this two-year study will produce a foundation of knowledge on a previously uninvestigated signal that enhances virulence gene expression in this important bacterial pathogen. This will add to our greater understanding of the spatial cues that govern the production of virulence factors by V. cholerae.